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The mechanisms underlying fetal lung injury remain poorly defined. MicroRNAs (miRNAs) are small noncoding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Using a nonhuman primate model of choriodecidual infection, we sought to determine if differentially expressed miRNAs were associated with acute fetal lung injury. After inoculating 10 chronically catheterized pregnant monkeys (Macaca nemestrina) with either group B streptococcus (GBS) at 1 × 10(6) CFU (n = 5) or saline (n = 5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled the changes in expression by microarray analysis. We identified 9 differentially expressed miRNAs in GBS-exposed fetal lungs, but of these, only miR-155-5p was validated by quantitative reverse transcription-PCR (P = 0.02). Significantly elevated miR-155-5p expression was also observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]). Overexpression of miR-155-5p in FeAE cells in turn increased the production of IL-6 and CXCL10/gamma interferon-induced protein 10, which are implicated in leukocyte recruitment but also in protection from lung injury. Interestingly, while miR-155-5p decreased fibroblast growth factor 9 (FGF9) expression in a luciferase reporter assay, FGF9 levels were actually increased in GBS-exposed fetal lungs in vivo. FGF9 overexpression is associated with abnormal lung development. Thus, upregulation of miR-155-5p may serve as a compensatory mechanism to lessen the increase in FGF9 and prevent aberrant lung development. Understanding the complicated networks regulating lung development in the setting of infection is a key step in identifying how to prevent fetal lung injury leading to bronchopulmonary dysplasia., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Intrauterine exposure to amniotic fluid (AF) cytokines is thought to predispose to bronchopulmonary dysplasia (BPD). We evaluated the effects of GBS exposure on RNA expression in fetal lung tissue to determine early molecular pathways associated with fetal lung injury that may progress to BPD., Methods: Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term = 172 days) received choriodecidual inoculation of either: 1) Group B Streptococcus (n = 5) or 2) saline (n = 5). Cesarean section and fetal necropsy was performed in the first week after GBS or saline inoculation regardless of labor. RNA was extracted from fetal lungs and profiled by microarray. Results were analyzed using single gene, Gene Set, and Ingenuity Pathway Analysis. Validation was by RT-PCR and immunohistochemistry., Results: Despite uterine quiescence in most cases, fetal lung injury occurred in four GBS cases (intra-alveolar neutrophils, interstitial thickening) and one control (peri-mortem hemorrhage). Significant elevations of AF cytokines (TNF-α, IL-8, IL-1β, IL-6) were detected in GBS versus controls (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable by culture and PCR in the AF and fetal lungs. A total of 335 genes were differentially expressed greater than 1.5 fold (p<0.05) with GBS exposure associated with a striking upregulation of genes in innate and adaptive immunity and downregulation of pathways for angiogenesis, morphogenesis, and cellular growth and development., Conclusions: A transient choriodecidual infection may induce fetal lung injury with profound alterations in the genetic program of the fetal lung before signs of preterm labor. Our results provide a window for the first time into early molecular pathways disrupting fetal lung angiogenesis and morphogenesis before preterm labor occurs, which may set the stage for BPD. A strategy to prevent BPD should target the fetus in utero to attenuate alterations in the fetal lung genetic program.

Activation of pulmonary defenses against Pseudomonas aeruginosa requires myeloid differentiation factor 88 (MyD88), an adaptor for Toll-like receptor (TLR) signaling. To determine which TLRs mediate recognition of P. aeruginosa, we measured cytokine responses of bone marrow cells from wild-type mice and mice lacking TLR2 (TLR2(-/-)), TLR4 (TLR4(-/-)), TLR2 and TLR4 (TLR2/4(-/-)), or MyD88 (MyD88(-/-)) to wild-type P. aeruginosa and to fliC P. aeruginosa, which lacks the TLR5 ligand flagellin. Mice also were challenged with aerosolized bacteria to determine cytokine responses, lung inflammation, and bacterial clearance. TNF induction required MyD88 and was absent in TLR2/4(-/-) cells in response to fliC but not wild-type P. aeruginosa, whereas TLR2(-/-) cells exhibited augmented responses. In vivo, TLR4(-/-) mice responded to wild-type P. aeruginosa with reduced cytokine production and inflammation, but intact bacterial clearance, while TLR2(-/-) mice had partially impaired cytokine responses and delayed bacterial killing despite normal inflammation. When challenged with fliC, MyD88(-/-) mice failed to mount early cytokine and inflammatory responses or control bacterial replication, resulting in necrotizing lung injury and lethal disseminated infection. TLR4(-/-) and TLR2/4(-/-) mice responded to fliC infection with severely limited inflammatory and cytokine responses but intact bacterial clearance. TLR2(-/-) mice had partially reduced cytokine responses but augmented inflammation and preserved bacterial killing. These data indicate that TLR4- and flagellin-induced signals mediate most of the acute inflammatory response to Pseudomonas and that TLR2 has a counterregulatory role. However, MyD88-dependent pathways, in addition to those downstream of TLR2, TLR4, and TLR5, are required for pulmonary defense against P. aeruginosa.

Purpose: The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys., Methods: Four female rhesus monkeys were dosed with lipid:DNA formulations via aerosol inhalation, where the DNA coded for the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein. Delivery of DNA was determined in lung samples by polymerase chain reaction (PCR) by qualitative and quantitative methods. Transgene specific messenger RNA was measured by reverse transcriptase PCR (RT-PCR) and protein expression and localization were evaluated by immunohistochemistry (IHC)., Results: Approximately four mg of DNA, complexed with cationic lipid 1.2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) and cholesterol were delivered to the lungs of animals by airjet nebulizer. Three days after dosing, tissue samples from the lung were collected and shown to have vector specific DNA, RNA and the presence of CFFR protein. Specifically, the hCFTR protein was distributed widely, although non-uniformly, throughout airway epithelium being located on the apical surface of epithelial cells. Importantly, no adverse clinical effects were observed and the lungs showed no histological abnormalities or signs of acute inflammation., Conclusions: This study shows that lipid:DNA formulations based on EDMPC and cholesterol can be administered to primates by nebulization resulting in measurable expression of the hCFTR protein. The absence of inflammation is also encouraging and such systems may have utility for delivery of genes to the lungs for the treatment of a variety of pulmonary diseases including cystic fibrosis.

TNF-alpha is a protein elaborated by monocytes and macrophages in response to endotoxin. The in vivo consequences of TNF-alpha elaboration have been examined extensively after intravenous administration of TNF-alpha. Substantially less is known about the effects of TNF-alpha that may be generated locally by resident tissue phagocytes. We investigated the direct effects of TNF-alpha on lung tissue by administering large amounts of human TNF-alpha directly to the lung, either as an aerosol or as an intratracheal bolus. Rats were exposed to an aerosol containing several concentrations of TNF-alpha, resulting in retention of significant quantities of TNF-alpha. The histologic response to inhaled TNF-alpha was characterized by adherence of leukocytes to venular endothelium, endothelial cell disruption, and bronchovascular edema. After aerosol administration, however, there was no evidence of alveolar inflammation or edema. In contrast, intravenous administration of large amounts of human TNF-alpha, at a dose that produced a lung content of TNF-alpha similar to that produced after high-concentration aerosol exposure, resulted in severe alveolar injury and edema. Intravenous administration of TNF-alpha did not result in the bronchovascular changes seen after inhalation. To ensure that sufficient quantities of TNF-alpha were being delivered to the lung, TNF-alpha was given as an intratracheal bolus to rats. This led to measurable absorption, but the spectrum and severity of lung injury was similar to the group that received TNF-alpha as an aerosol. We conclude that in rats, the pulmonary response to the injurious effects of TNF-alpha differ, depending on whether the TNF-alpha is delivered to the air or blood side of the alveolar capillary barrier.

Alveolar macrophages were obtained from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and domestic sheep for the purpose of comparing pulmonary host defense mechanisms in the two species. Specific variables studied included (1) characterization of the cell types present in the lung, (2) alveolar macrophage phagocytic and bactericidal functions, (3) measurement of protein levels in lavage fluid, and (4) measurement of cortisol levels in lavage fluid. While phagocytic cell populations were similar between bighorn and domestic sheep, a significantly higher percentage of lymphocytes were present in bighorns than domestics (20% in bighorn versus 6% in domestic sheep). Significant differences were not observed in the phagocytic or bactericidal functions of macrophages between the two species. Significant differences were not observed in either lavage fluid protein levels or in cortisol levels.

The purpose of this article is to examine factors contributing to defense of the bovine lung from microbial infection. Appropriate physical, cellular, and secretory defense components are assessed. Attention is paid to the thin line separating host-mediated defense from host-mediated injury of the lung.